The present invention relates to bicyclic heterocycles of general formula
the tautomers, the stereoisomers, the mixtures and the salts thereof, particularly the physiologically acceptable salts thereof with inorganic or organic acids or bases which have valuable pharmacological properties, particularly an inhibitory effect on signal transduction mediated by tyrosine kinases, the use thereof for treating diseases, particularly tumoral diseases, as well as benign prostatic hyperplasia (BPH), diseases of the lungs and respiratory tract, and the preparation thereof.
In the above general formula I
Ra denotes a hydrogen atom or a C1-4-alkyl group,
Rb denotes a phenyl, benzyl or 1-phenylethyl group, wherein the phenyl nucleus is substituted in each case by the groups R1 to R3, while
                R1 and R2, which may be identical or different, in each case denote a hydrogen, fluorine, chlorine, bromine or iodine atom,        a C1-4-alkyl, hydroxy, C1-4-alkoxy, C2-3-alkenyl or C2-3-alkynyl group,        an aryl, aryloxy, arylmethyl or arylmethoxy group,        a heteroaryl, heteroaryloxy, heteroarylmethyl or heteroarylmethoxy group,        a methyl or methoxy group substituted by 1 to 3 fluorine atoms or        a cyano, nitro or amino group, and        R3 denotes a hydrogen, fluorine, chlorine or bromine atom,        a methyl or trifluoromethyl group,Rc denotes a hydrogen atom or a fluorine, chlorine or bromine atom,a hydroxy or C1-4-alkyloxy group,a methoxy group substituted by 1 to 3 fluorine atoms,an ethyloxy group substituted by 1 to 5 fluorine atoms,a C2-4-alkyloxy group which is substituted by a group R4, where        R4 denotes a hydroxy, C1-3-alkyloxy, C3-6-cycloalkyloxy, C3-6-cycloalkyl-C1-3-alkyloxy, amino, C1-3-alkylamino, di-(C1-3-alkyl)amino, bis-(2-C1-3-alkyloxy-ethyl)-amino, bis-(3-C1-3-alkyloxy-propyl)-amino, pyrrolidin-1-yl, piperidin-1-yl, homopiperidin-1-yl, morpholin-4-yl, homomorpholin-4-yl, piperazin-1-yl, 4-(C1-3-alkyl)-piperazin-1-yl, homopiperazin-1-yl or 4-(C1-3-alkyl)-homopiperazin-1-yl group,a C2-4-alkyloxy group which is substituted by the group E, where E is defined as hereinafter,a C3-7-cycloalkyloxy or C3-7-cycloalkyl-C1-4-alkyloxy group,a tetrahydrofuran-3-yloxy, tetrahydropyran-3-yloxy or tetrahydropyran-4-yloxy group,a tetrahydrofuranyl-C1-4-alkyloxy or tetrahydropyranyl-C1-4-alkyloxy group,a pyrrolidin-3-yloxy, piperidin-3-yloxy or piperidin-4-yloxy-group,a 1-(C1-3-alkyl)-pyrrolidin-3-yloxy, 1-(C1-3-alkyl)-piperidin-3-yloxy or 1-(C1-3-alkyl)-piperidin-4-yloxy-group,a C1-4-alkoxy group which is substituted by a pyrrolidinyl, piperidinyl or homopiperidinyl group substituted in the 1 position by the group R5, where        R5 denotes a hydrogen atom or a C1-3-alkyl group,or a C1-4-alkoxy group which is substituted by a morpholinyl or homomorpholinyl group substituted in the 4 position by the group R5,A denotes an imino or C1-4-alkylimino group,B denotes a carbonyl or sulphonyl group,C denotes a 1,3-allenylene, 1,1-vinylene or 1,2-vinylene group which may be substituted in each case by one or two methyl groups or by a trifluoromethyl group, an ethynylene group ora 1,3-butadien-1,4-ylene group which may be substituted by one or two methyl groups or by a trifluoromethyl group,D denotes a straight-chain or branched C1-4-alkylene group,E denotes a pyrrolidin-1-yl group wherein two hydrogen atoms on the carbon skeleton are replaced by a straight-chain alkylene bridge, this bridge containing 2 to 6 carbon atoms if the two hydrogen atoms are located on the same carbon atom, or 1 to 5 carbon atoms if the two hydrogen atoms are located on adjacent carbon atoms, or 2 to 4 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by one atom, while said pyrrolidin-1-yl groups may each additionally be substituted by one or two C1-3-alkyl groups,a piperidin-1-yl or homopiperidin-1-yl group, wherein in each case two hydrogen atoms on the carbon skeleton are replaced by a straight-chain alkylene bridge, this bridge containing 2 to 6 carbon atoms if the two hydrogen atoms are located on the same carbon atom, or 1 to 5 carbon atoms if the two hydrogen atoms are located on adjacent carbon atoms, or 1 to 4 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by one atom, or 1 to 3 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by two atoms, while said piperidin-1-yl- and homopiperidin-1-yl groups may each additionally be substituted by one or two C1-3-alkyl groups,a piperazin-1-yl or homopiperazin-1-yl group, wherein in each case two hydrogen atoms on the carbon skeleton are replaced by a straight-chain alkylene bridge, this bridge containing 2 to 6 carbon atoms if the two hydrogen atoms are located on the same carbon atom, or 1 to 5 carbon atoms if the two hydrogen atoms are located on adjacent carbon atoms, or 1 to 4 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by one atom, or 1 to 3 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by two atoms, while said piperazin-1-yl- and homopiperazin-1-yl groups may each additionally be substituted by one or two C1-3-alkyl groups,a morpholin-4-yl or homomorpholin-4-yl group, wherein in each case two hydrogen atoms on the carbon skeleton are replaced by a straight-chain alkylene bridge, this bridge containing 2 to 6 carbon atoms if the two hydrogen atoms are located on the same carbon atom, or 1 to 5 carbon atoms if the two hydrogen atoms are located on adjacent carbon atoms, or 1 to 4 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by one atom, or 1 to 3 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by two atoms, while said morpholin-4-yl- and homomorpholin-4-yl may each additionally be substituted by one or two C1-3-alkyl groups,andX denotes a methyne group substituted by a cyano group or a nitrogen atom,whilst by the aryl groups mentioned in the definition of the above groups is meant in each case a phenyl group which is mono- or disubstituted by R6, while the substituents may be identical or different and        R6 denotes a hydrogen atom, a fluorine, chlorine, bromine or iodine atom or a C1-3-alkyl, hydroxy, C1-3-alkyloxy, difluoromethyl, trifluoromethyl, difluoromethoxy, trifluoromethoxy or cyano group,by the heteroaryl groups mentioned in the definition of the above groups is meant a pyridyl, pyridazinyl, pyrimidinyl or pyrazinyl group, while said heteroaryl groups are each mono- or disubstituted by the group R6, while the substituents may be identical or different and R6 is as hereinbefore defined, andunless otherwise stated, said alkyl groups may be straight-chained or branched.        
Preferred compounds of the above general formula I are those wherein
Ra denotes a hydrogen atom,
Rb denotes a phenyl group substituted by the groups R1 to R3, where
                R1 denotes a hydrogen, fluorine, chlorine or bromine atom,        a methyl, trifluoromethyl or ethynyl group,        a phenyloxy or phenylmethoxy group, wherein the phenyl moiety of said groups is optionally substituted by a fluorine or chlorine atom, or        a pyridinyloxy or pyridinylmethoxy group, wherein the pyridinyl moiety of said groups is optionally substituted by a methyl or trifluoromethyl group,        R2 denotes a hydrogen, fluorine or chlorine atom and        R3 denotes a hydrogen atom,Rc denotes a hydrogen atom,a C1-3-alkyloxy group,a C4-6-cycloalkyloxy or C3-6-cycloalkyl-C1-2-alkyloxy-group,a tetrahydrofuran-3-yloxy, tetrahydropyran-3-yloxy, tetrahydropyran-4-yloxy, tetrahydrofuranyl-C1-2-alkyloxy or tetrahydropyranyl-C1-2-alkyloxy group,an ethyloxy group which is substituted in the 2 position by a group R4, where        R4 denotes a hydroxy, C1-3-alkyloxy, amino, C1-3-alkylamino, di-(C1-3-alkyl)amino, bis-(2-methoxyethyl)-amino, pyrrolidin-1-yl, piperidin-1-yl, homopiperidin-1-yl, morpholin-4-yl, homomorpholin-4-yl, piperazin-1-yl, 4-(C1-3-alkyl)-piperazin-1-yl, homopiperazin-1-yl, or 4-(C1-3-alkyl)-homopiperazin-1-yl group,a propyloxy group which is substituted in the 3 position by a group R4, where R4 is as hereinbefore defined, ora butyloxy group which is substituted in the 4 position by a group R4 where R4 is as hereinbefore defined,A denotes an imino group,B denotes a carbonyl or sulphonyl group,C denotes a 1,1-vinylene, 1,2-vinylene or ethynylene group,D denotes a methylene, 1,1-ethylene or 1,2-ethylene group,E denotes a piperidin-1-yl group wherein two hydrogen atoms on the carbon skeleton are replaced by a straight-chain alkylene bridge, this bridge containing 2 to 5 carbon atoms if the two hydrogen atoms are located on the same carbon atom, or 1 to 4 carbon atoms if the two hydrogen atoms are located on adjacent carbon atoms, or 1 to 3 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by one atom, or 1 or 2 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by two atoms, while said piperidin-1-yl groups may each additionally be substituted by one or two C1-3-alkyl groups,a piperazin-1-yl group wherein two hydrogen atoms on the carbon skeleton are replaced by a straight-chain alkylene bridge, this bridge containing 2 to 5 carbon atoms if the two hydrogen atoms are located on the same carbon atom, or 1 to 4 carbon atoms if the two hydrogen atoms are located on adjacent carbon atoms, or 1 to 3 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by one atom, or 1 or 2 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by two atoms, while said piperazin-1-yl groups may each additionally be substituted by one or two C1-3-alkyl groups, ora morpholin-4-yl group wherein two hydrogen atoms on the carbon skeleton are replaced by a straight-chain alkylene bridge, this bridge containing 2 to 5 carbon atoms if the two hydrogen atoms are located on the same carbon atom, or 1 to 4 carbon atoms if the two hydrogen atoms are located on adjacent carbon atoms, or 1 to 3 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by one atom, or 1 or 2 carbon atoms if the two hydrogen atoms are located on carbon atoms which are separated by two atoms, while said morpholin-4-yl groups may each additionally be substituted by one or two C1-3-alkyl groups,andX represents a nitrogen atom,while, unless otherwise stated, said alkyl groups may be straight-chain or branched,their tautomers, their stereoisomers, their mixtures and their salts.        
Particularly preferred compounds of the above general formula I are those wherein
Ra denotes a hydrogen atom,
Rb denotes a 3-ethynylphenyl, 3-bromophenyl, 3,4-difluorophenyl or 3-chloro-4-fluoro-phenyl group,
Rc denotes a hydrogen atom,
a methoxy, ethyloxy, 2-(methoxy)ethyloxy, 3-(morpholin-4-yl)propyloxy, cyclo-butyloxy, cyclopentyloxy, cyclohexyloxy, cyclopropylmethoxy, cyclopentylmethoxy, tetrahydrofuran-3-yloxy, tetrahydropyran-3-yloxy, tetrahydropyran-4-yloxy, tetrahydrofuran-2-yl methoxy, tetrahydrofuran-3-yl methoxy or tetrahydropyran-4-yl-methoxy group,A denotes an imino group,B denotes a carbonyl group,C denotes a 1,2-vinylene group,D denotes a methylene group,E denotes a 2-aza-bicyclo[2.2.1]hept-2-yl, 2,5-diaza-bicyclo[2.2.1]hept-2-yl, 5-methyl-2,5-diaza-bicyclo[2.2.1]hept-2-yl, 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl, 2-aza-bicyclo[2.2.2]oct-2-yl, 3-aza-bicyclo[3.2.1]oct-3-yl, 8-aza-bicyclo[3.2.1]oct-8-yl, 3,8-diaza-bicyclo[3.2.1]oct-3-yl, 8-methyl-3,8-diaza-bicyclo[3.2.1]oct-3-yl, 3,8-diaza-bicyclo[3.2.1]oct-8-yl, 3-methyl-3,8-diaza-bicyclo[3.2.1]oct-8-yl, 3-oxa-8-aza-bicyclo[3.2.1]oct-8-yl or 8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl group andX denotes a nitrogen atom,their tautomers, their stereoisomers, their mixtures and their salts.
Most particularly preferred compounds of general formula I are those wherein
Ra denotes a hydrogen atom,
Rb denotes a 3-chloro-4-fluoro-phenyl group,
Rc denotes a tetrahydrofuran-3-yloxy, cyclopentyloxy or cyclopropylmethoxy group,
A denotes an imino group,
B denotes a carbonyl group,
C denotes a 1,2-vinylene group,
D denotes a methylene group,
E denotes a 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl group, a 3-oxa-8-aza-bicyclo[3.2.1]oct-8-yl group or an 8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl group
and
X denotes a nitrogen atom,
their tautomers, their stereoisomers, their mixtures and their salts.
The following are mentioned as examples of particularly preferred compounds of general formula I:    (a) 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-((1S,4S)-2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl)-1-oxo-2-buten-1-yl]amino}-7-cyclopropylmethoxy-quinazoline,    (b) 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-((1S,4S)-2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl)-1-oxo-2-buten-1-yl]amino}-7-cyclopentyloxy-quinazoline,    (c) 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-((1S,4S)-2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,    (d) 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(3-oxa-8-aza-bicyclo[3.2.1]oct-8-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,    (e) 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-((1R,4R)-2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,    (f) 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,    (g) 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-((1R,4R)-2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl)-1-oxo-2-buten-1-yl]amino}-7-[(R)-(tetrahydrofuran-3-yl)oxy]-quinazoline and    (h) 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-((1S,4S)-2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl)-1-oxo-2-buten-1-yl]amino}-7-[(R)-(tetrahydrofuran-3-yl)oxy]-quinazoline,and the salts thereof.
The compounds of general formula I may be prepared for example by the following methods:
a) reacting a compound of general formula
whereinRa, Rb, Rc, A, B and X are as hereinbefore defined and R7 and R8, which may be identical or different, denote C1-4-alkyl groups,with a compound of general formulaOHC-D-E,  (III)whereinD and E are as hereinbefore defined.
The reaction is expediently carried out in a solvent or mixture of solvents such as tetrahydrofuran, tetrahydrofuran/water, acetonitrile, acetonitrile/water, dioxane, ethyleneglycol dimethyl ether, isopropanol, methylene chloride, dimethylformamide or sulpholane optionally in the presence of an inorganic or organic base, e.g. sodium carbonate, potassium hydroxide or 1,8-diazabicyclo[5.4.0]undec-7-ene and optionally in the presence of a lithium salt such as lithium chloride at temperatures between −50 and 150° C., but preferably at temperatures between −20 and 80° C. The reaction may also be carried out with a reactive derivative of the compound of general formula III, for example the hydrate or a hemiacetal.
b) reacting a compound of general formula
wherein Ra, Rb, Rc, A, B, C, D and X are as hereinbefore defined and Z1 denotes a leaving group such as a halogen atom or a substituted sulphonyloxy group such as a chlorine or bromine atom, a methanesulphonyloxy or p-toluenesulphonyloxy group,with a compound of general formulaH-E,  (V)wherein E is as hereinbefore defined.
The reaction is expediently carried out in a solvent such as isopropanol, butanol, tetrahydrofuran, dioxane, acetonitrile, dimethylformamide, sulpholane, toluene or methylene chloride or mixtures thereof, optionally in the presence of an inorganic or organic base, e.g. sodium carbonate, potassium carbonate, potassium hydroxide, triethylamine or N-ethyl-diisopropylamine and optionally in the presence of a reaction accelerator such as an alkali metal iodide at temperatures between −20 and 150° C., but preferably at temperatures between 0 and 100° C. The reaction may, however, also be carried out without a solvent or in an excess of the compound of general formula V used.
In the reactions described hereinbefore, any reactive groups present such as hydroxy, amino, alkylamino or imino groups may be protected during the reaction by conventional protecting groups which are cleaved again after the reaction.
For example, a protecting group for a hydroxy group may be a trimethylsilyl, acetyl, trityl, benzyl or tetrahydropyranyl group.
Protecting groups for an amino, alkylamino or imino group may be a formyl, acetyl, trifluoroacetyl, ethoxycarbonyl, tert.butoxycarbonyl, benzyloxycarbonyl, benzyl, methoxybenzyl or 2,4-dimethoxybenzyl group, for example.
Any protecting group used is optionally subsequently cleaved for example by hydrolysis in an aqueous solvent, e.g. in water, isopropanol/water, acetic acid/water, tetrahydrofuran/water or dioxan/water, in the presence of an acid such as trifluoroacetic acid, hydrochloric acid or sulphuric acid or in the presence of an alkali metal base such as sodium hydroxide or potassium hydroxide or aprotically, e.g. in the presence of iodotrimethylsilane, at temperatures between 0 and 120° C., preferably at temperatures between 10 and 100° C.
However, a benzyl, methoxybenzyl or benzyloxycarbonyl group is cleaved, for example hydrogenolytically, e.g. with hydrogen in the presence of a catalyst such as palladium/charcoal in a suitable solvent such as methanol, ethanol, ethyl acetate or glacial acetic acid, optionally with the addition of an acid such as hydrochloric acid at temperatures between 0 and 100° C., but preferably at ambient temperatures between 20 and 60° C., and at a hydrogen pressure of 1 to 7 bar, but preferably 3 to 5 bar. A 2,4-dimethoxybenzyl group, however, is preferably cleaved in trifluoroacetic acid in the presence of anisole.
A tert.butyl or tert.butyloxycarbonyl group is preferably cleaved by treating with an acid such as trifluoroacetic acid or hydrochloric acid or by treating with iodotrimethylsilane optionally using a solvent such as methylene chloride, dioxan, methanol or diethyl ether.
A trifluoroacetyl group is preferably cleaved by treating with an acid such as hydrochloric acid, optionally in the presence of a solvent such as acetic acid at temperatures between 50 and 120° C. or by treating with sodium hydroxide solution, optionally in the presence of a solvent such as tetrahydrofuran at temperatures between 0 and 50° C.
Moreover, the compounds of general formula I obtained may be resolved into their enantiomers and/or diastereomers, as mentioned hereinbefore. Thus, for example, cis/trans mixtures may be resolved into their cis and trans isomers, and compounds with at least one optically active carbon atom may be separated into their enantiomers.
Thus, for example, the cis/trans mixtures may be resolved by chromatography into the cis and trans isomers thereof, the compounds of general formula I obtained which occur as racemates may be separated by methods known per se (cf. Allinger N. L. and Eliel E. L. in “Topics in Stereochemistry”, Vol. 6, Wiley Interscience, 1971) into their optical antipodes and compounds of general formula I with at least 2 asymmetric carbon atoms may be resolved into their diastereomers on the basis of their physical-chemical differences using methods known per se, e.g. by chromatography and/or fractional crystallisation, and, if these compounds are obtained in racemic form, they may subsequently be resolved into the enantiomers as mentioned above.
The enantiomers are preferably separated by column separation on chiral phases or by recrystallisation from an optically active solvent or by reacting with an optically active substance which forms salts or derivatives such as e.g. esters or amides with the racemic compound, particularly acids and the activated derivatives or alcohols thereof, and separating the diastereomeric mixture of salts or derivatives thus obtained, e.g. on the basis of their differences in solubility, whilst the free antipodes may be released from the pure diastereomeric salts or derivatives by the action of suitable agents. Optically active acids in common use are e.g. the D- and L-forms of tartaric acid or dibenzoyltartaric acid, di-o-tolyltartaric acid, malic acid, mandelic acid, camphorsulphonic acid, glutamic acid, aspartic acid or quinic acid. An optically active alcohol may be for example (+) or (−)-menthol and an optically active acyl group in amides may be a (+)- or (−)-menthyloxycarbonyl, for example.
Furthermore, the compounds of formula I may be converted into the salts thereof, particularly for pharmaceutical use into the physiologically acceptable salts with inorganic or organic acids. Acids which may be used for this purpose include for example hydrochloric acid, hydrobromic acid, sulphuric acid, methanesulphonic acid, phosphoric acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid or maleic acid.
As already mentioned hereinbefore, the compounds of general formula I according to the invention and the physiologically acceptable salts thereof have valuable pharmacological properties, particularly an inhibiting effect on signal transduction mediated by the Epidermal Growth Factor receptor (EGF-R), whilst this may be achieved for example by inhibiting ligand bonding, receptor dimerisation or tyrosine kinase itself. It is also possible that the transmission of signals to components located downstream is blocked.
The biological properties of the new compounds were investigated as follows:
The inhibition of human EGF-receptor kinase was determined using the cytoplasmic tyrosine kinase domain (methionine 664 to alanine 1186 based on the sequence published in Nature 309 (1984), 418). For this the protein was expressed in Sf9 insect cells as GST fusion protein using the Baculovirus expression system.
The enzyme activity was measured in the presence or absence of the test compounds in serial dilutions. The polymer pEY (4:1) obtained from SIGMA was used as the substrate. Biotinylated pEY (bio-pEY) was added as the tracer substrate. 100 μl of reaction solution contained 10 μl of the inhibitor in 50% DMSO, 20 μl of the substrate solution (200 mM HEPES pH 7.4, 50 mM magnesium acetate, 2.5 mg/ml poly(EY), 5 μg/ml bio-pEY) and 20 μl of enzyme preparation. The enzyme reaction was started by the addition of 50 μl of a 100 μM ATP solution in 10 mM of magnesium chloride. The dilution of the enzyme preparation was adjusted so that the incorporation of phosphate in the bio-pEY was linear in terms of time and quantity of enzyme. The enzyme preparation was diluted in 20 mM HEPES pH 7.4, 1 mM EDTA, 130 mM common salt, 0.05% Triton X-100, 1 mM DTT and 10% glycerol.
The enzyme assays were carried out at ambient temperature over a period of 30 minutes and ended by the addition of 50 μl of a stopping solution (250 mM EDTA in 20 mM HEPES pH 7.4). 100 μl were placed on a streptavidin-coated microtitre plate and incubated for 60 minutes at ambient temperature. Then the plate was washed with 200 μl of a wash solution (50 mM Tris, 0.05% Tween 20). After the addition of 100 μl of an HRPO-labelled anti-PY antibody (PY20H Anti-PTyr:HRP made by Transduction Laboratories, 250 ng/ml) the preparation was incubated for 60 minutes. Then the microtitre plate was washed three times with 200 μl of wash solution. The samples were then combined with 100 μl of a TMB-peroxidase solution (A:B=1:1, Kirkegaard Perry Laboratories). After 10 minutes the reaction was stopped. The extinction was measured at OD450nm with an ELISA reader. All the results were measured three times.
The data were adapted by iterative calculation using an analytical programme for sigmoid curves (Graph Pad Prism Version 3.0) with a variable Hill pitch. All the iterative data produced had a correlation coefficient of more than 0.9 and the upper and lower values of the curves showed a spread of at least a factor of 5. The active substance concentration which inhibits the activity of EGF receptor kinase by 50% (IC50) was derived from the curves.
The following results are shown in Table I:
TABLE IInhibition of EGF-receptorCompoundkinase(Example No.)IC50 [nM]11.51(1)0.520.52(2)0.52(3)2
The compounds of general formula I according to the invention thus inhibit signal transduction by tyrosine kinases, as demonstrated by the example of the human EGF receptor, and are therefore useful for treating pathophysiological processes caused by hyperfunction of tyrosine kinases. These are e.g. benign or malignant tumours, particularly tumours of epithelial and neuroepithelial origin, metastasisation and the abnormal proliferation of vascular endothelial cells (neoangiogenesis).
The compounds according to the invention are also useful for preventing and treating diseases of the airways and lungs which are accompanied by increased or altered production of mucus caused by stimulation by tyrosine kinases, e.g. in inflammatory diseases of the airways such as chronic bronchitis, chronic obstructive bronchitis, asthma, bronchiectasis, allergic or non-allergic rhinitis or sinusitis, cystic fibrosis, α1-antitrypsin deficiency, or coughs, pulmonary emphysema, pulmonary fibrosis and hyperreactive airways.
The compounds are also suitable for treating diseases of the gastrointestinal tract and bile duct and gall bladder which are associated with disrupted activity of the tyrosine kinases, such as may be found e.g. in chronic inflammatory changes such as cholecystitis, Crohn's disease, ulcerative colitis, and ulcers in the gastrointestinal tract or such as may occur in diseases of the gastrointestinal tract which are associated with increased secretions, such as Ménétrier's disease, secreting adenomas and protein loss syndrome.
In addition, the compounds of general formula I and the physiologically acceptable salts thereof may be used to treat other diseases caused by abnormal function of tyrosine kinases, such as e.g. epidermal hyperproliferation (psoriasis), benign prostatic hyperplasia (BPH), inflammatory processes, diseases of the immune system, hyperproliferation of haematopoietic cells, etc.
By reason of their biological properties the compounds according to the invention may be used on their own or in conjunction with other pharmacologically active compounds, for example in tumour therapy, in monotherapy or in conjunction with other anti-tumour therapeutic agents, for example in combination with topoisomerase inhibitors (e.g. etoposide), mitosis inhibitors (e.g. vinblastine), compounds which interact with nucleic acids (e.g. cis-platin, cyclophosphamide, adriamycin), hormone antagonists (e.g. tamoxifen), inhibitors of metabolic processes (e.g. 5-FU etc.), cytokines (e.g. interferons), antibodies, etc. For treating respiratory tract diseases, these compounds may be used on their own or in conjunction with other therapeutic agents for the airways, such as substances with a secretolytic (e.g. ambroxol, N-acetylcysteine), broncholytic (e.g. tiotropium or ipratropium or fenoterol, salmeterol, salbutamol) and/or anti-inflammatory activity (e.g. theophylline or glucocorticoids). For treating diseases in the region of the gastrointestinal tract, these compounds may also be administered on their own or in conjunction with substances having an effect on motility or secretion. These combinations may be administered either simultaneously or sequentially.
These compounds may be administered either on their own or in conjunction with other active substances by intravenous, subcutaneous, intramuscular, intraperitoneal or intranasal route, by inhalation or transdermally or orally, whilst aerosol formulations are particularly suitable for inhalation.
For pharmaceutical use the compounds according to the invention are generally used for warm-blooded vertebrates, particularly humans, in doses of 0.01-100 mg/kg of body weight, preferably 0.1-15 mg/kg. For administration they are formulated with one or more conventional inert carriers and/or diluents, e.g. with corn starch, lactose, glucose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid, water, water/ethanol, water/glycerol, water/sorbitol, water/polyethylene glycol, propylene glycol, stearyl alcohol, carboxymethylcellulose or fatty substances such as hard fat or suitable mixtures thereof in conventional galenic preparations such as plain or coated tablets, capsules, powders, suspensions, solutions, sprays or suppositories.
The following Examples are intended to illustrate the present invention without restricting it:
Preparation of the Starting Compounds: